Mapping Tumor Spheroid Mechanics in Dependence of 3D Microenvironment Stiffness and Degradability by Brillouin Microscopy

Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids’ cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids’ mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.

Mapping tumor spheroid mechanics in dependence of 3D microenvironment stiffness and degradability by Brillouin microscopy

Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)- degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.

Coaxially Bioprinted Cell-Laden Tubular-Like Structure for Studying Glioma Angiogenesis

Glioblastomas are the most frequently diagnosed and one of the most lethal primary brain tumors, and one of their key features is a dysplastic vascular network. However, because the origin of the tumor blood vessels remains controversial, an optimal preclinical tumor model must be established to elucidate the tumor angiogenesis mechanism, especially the role of tumor cells themselves in angiogenesis. Therefore, shell-glioma cell (U118)-red fluorescent protein (RFP)/core-human umbilical vein endothelial cell (HUVEC)-green fluorescent protein (GFP) hydrogel microfibers were coaxially bioprinted. U118–RFP and HUVEC–GFP cells both exhibited good proliferation in a three-dimensional (3D) microenvironment. The secretability of both vascular endothelial growth factor A and basic fibroblast growth factor was remarkably enhanced when both types of cells were cocultured in 3D models. Moreover, U118 cells promoted the vascularization of the surrounding HUVECs by secreting vascular growth factors. More importantly, U118–HUVEC-fused cells were found in U118–RFP/HUVEC–GFP hydrogel microfibers. Most importantly, our results indicated that U118 cells can not only recruit the blood vessels of the surrounding host but also directly transdifferentiate into or fuse with endothelial cells to participate in tumor angiogenesis in vivo. The coaxially bioprinted U118–RFP/HUVEC–GFP hydrogel microfiber is a model suitable for mimicking the glioma microenvironment and for investigating tumor angiogenesis.

3D Microenvironment-Specific Mechanosensing Regulates Neural Stem Cell Lineage Commitment

While extracellular matrix (ECM) mechanics strongly regulate stem cell commitment, the field′s mechanistic understanding of this phenomenon largely derives from simplified two–dimensional (2D) culture substrates. Here we found a three-dimensional (3D) matrix–specific mechanoresponsive mechanism for neural stem cell (NSC) differentiation. NSC lineage commitment in 3D is maximally stiffness-sensitive in the range of 0.1–1.2 kPa, a narrower and more brain-mimetic range than we had previously identified in 2D (0.75–75 kPa). Transcriptomics revealed stiffness-dependent upregulation of early growth response 1 (Egr1) in 3D but not in 2D. Egr1 knockdown enhanced neurogenesis in stiff ECMs by driving β–catenin nuclear localization and activity in 3D, but not in 2D. Mechanical modeling and experimental studies under osmotic pressure indicate that stiff 3D ECMs are likely to stimulate Egr1 via increases in confining stress during cell volumetric growth. To our knowledge, Egr1 represents the first 3D–specific stem cell mechanoregulatory factor.

Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells

The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.

Design and Fabrication of Microfluidic-Based 3D Microphysiological Systems for Studying Cell Migration and Invasion Behaviors

The cell migration and invasion behaviors play pivotal roles in tissue regeneration. For the skin repair process, a directed inflammatory response that regulates fibroblasts is critical for efficient wound healing. In this study, the authors present the design and fabrication of a microfluidic-based three-dimensional (3D) microphysiological system and how it impacts in controlling fibroblast migration and invasion under the induction of differently polarized macrophages. The microfluidic device had two chambers on opposite sides of a 1 mm micochannel, providing directed induction and sufficient width for long-term observation. The test cells could be seeded with or without matrix gel, cultured in a 2D or 3D microenvironment according to experiment settings. The microchannel allowed for any sorts of matrix filling and was on-demanding for continuous surveillance. Herein, our microfluidic device reserved the advantages of traditional methods using transwell chamber or scratch wound healing assay. In addition, it even came with more superiority such as retrievability, dynamic observation, and 3D environment simulation. The migration and invasion pattern of NIH3T3 modulated by RAW264.7 macrophages in different polarization status was demonstrated as an example. The results of the migration assay corresponded with that of the proliferation and gene expression experiments, verifying that our device was fully capable of restoring in vivo microenvironment and presenting cellular motility behaviors.

Three-dimensional microenvironment regulates gene expression, function, and tight junction dynamics of iPSC-derived blood-brain barrier microvessels

The blood-brain barrier (BBB) plays a pivotal role in brain health and disease. In the BBB, brain microvascular endothelial cells (BMECs) are connected by tight junctions which regulate paracellular transport, and express specialized transporter systems which regulate transcellular transport. However, existing in vitro models of the BBB display variable physiological accuracy across a wide range of characteristics including gene/protein expression and barrier function. Here, we use an isogenic family of fluorescently-labeled iPSC-derived BMEC-like cells (iBMECs) and brain pericyte-like cells (iPCs) within two-dimensional confluent monolayers (2D) and three-dimensional (3D) tissue-engineered microvessels to explore how 3D microenvironment regulates gene expression and function of the in vitro BBB. We show that 3D microenvironment (shear stress, cell-ECM interactions, and cylindrical geometry) increases BBB phenotype and endothelial identity, and alters angiogenic and cytokine responses in synergy with pericyte co-culture. Tissue-engineered microvessels incorporating junction-labeled iBMECs enable study of the real-time dynamics of tight junctions during homeostasis and in response to physical and chemical perturbations.

Engineering and Monitoring 3D Cell Constructs with Time-Evolving Viscoelasticity for the Study of Liver Fibrosis In Vitro

Liver fibrosis is generally associated with an over-production and crosslinking of extracellular matrix proteins, causing a progressive increase in both the elastic and viscous properties of the hepatic tissue. We describe a strategy for mimicking and monitoring the mechano-dynamics of the 3D microenvironment associated with liver fibrosis. Cell-laden gelatin hydrogels were crosslinked with microbial transglutaminase using a purpose-designed cytocompatible two-step protocol, which allows for the exposure of cells to a mechanically changing environment during culturing. A bioreactor was re-engineered to monitor the mechanical properties of cell constructs over time. The results showed a shift towards a more elastic (i.e., solid-like) behaviour, which is likely related to an increase in cell stress. The method effectively mimics the time-evolving mechanical microenvironment associated with liver fibrosis and could provide novel insights into pathophysiological processes in which both elastic and viscous properties of tissues change over time.